PCR L8

Question 1

What 2 things does RT-PCR stand for?

Answer 1

Reverse-transcriptase PCR

Real time PCR (older term not used)

Question 2

What does qPCR mean?

Answer 2

Real time quantitative PCR

Question 3

What does RT-qPCR mean?

Answer 3

Reverse-transcriptase quantitative PCR.

Question 4

If a curve on an amplification curve graph rises faster than the other curves, what does this mean?

Answer 4

It indicates that the sample had a higher concentration of the virus.

Question 5

What does qPCR monitor?

Answer 5

DNA amplification as the reaction progresses by an increase of the fluorescence as double stranded DNA is formed.

Question 6

When there are double strands of DNA in the PCR will the fluorescence be strong or weak?

Answer 6

Strong

Question 7

When there are single strands of DNA will the fluorescence be strong or weak?

Answer 7

Weak

Question 8

What are the 4 distinct features of a qPCR curve?

Answer 8

LAG, exponential, linear and plateau.

Question 9

What are the two detection formats used for qPCR?

Answer 9

Non-specific dyes

Labelled probes

Question 10

What detection format for qPCR involves SYBR green dye that binds to all ouble stranded DNA molecules, regardless of the sequence. Fluorescence increases when the dye is intercalated into dsDNA. The intensity of fluorescence is dependent on the amount of dsDNA in the tube.

Answer 10

Non-specific dyes.

Question 11

What detection format for qPCR involves fluorescent molecules attached to a DNA probe, complementary to a region between two PCR primers on the target DNA. Another molecule attached to the probe is a quencher and it extinguishes the fluorescence when they’re in close proximity. During DNA synthesis when the primer reaches the probe, it is hydrolyzed/destroyed. This leads to the flurofor to be separated from the quencher- generating more fluorescence.

Answer 11

Labelled probes

Question 12

If the curve on a quantification cycle crosses the threshold at the cycle number 20, then what is the value of Cq?

Answer 12

20

Question 13

On a quantification cycle graph, when is a sample considered positive?

Answer 13

When it crosses the threshold.

Question 14

What does the Cq represent?

Answer 14

The point when the sample crosses the threshold and becomes positive.

Question 15

What does it mean if there is a large Cq value?

Answer 15

There is lower amounts of virus present - it took longer amount of time to cross the threshold.

Question 16

What efficiency for a standard curve is acceptible?

Answer 16

90-100%

Question 17

What is on the y axis and x axis of the Cq Standard Curve?

Answer 17

Y- Cq Values

X- Quantity of viral DNA

Question 18

What does the standard curve tell us?

Answer 18

How well a given PCR assay performed.

Question 19

What does the R-squared value of the standard curve tell us?

Answer 19

How close the data is to the fitted regression line. It should be as close as possible to 1 for a good PCR assay.

Question 20

What does the efficiency of the standard curve indicate?

Answer 20

How good the assay is at duplicating the specific target in each subsequent cycle within the exponential phase of the assay.

Question 21

What can you assess with the standard curve?

Answer 21

The level of viral nucleic acid in the sample.

Question 22

What factors influence at what temperature the double stranded DNA separates at?

Answer 22

Length and composition (A, T, C, G)

Question 23

When is melting analysis performed?

Answer 23

At the end of the PCR if the sample is positive.

Question 24

Should the temperature be raised slowly or quickly during a melting analysis?

Answer 24

Slowly

Question 25

What happens when the DNA strands begin to separate during the melting analysis?

Answer 25

Loss of fluorescence.

Question 26

If the negative control sample (water) appears positive in a melting analysis- how would you interpret the result?

Answer 26

False positive (non-specific amplification). The peak displayed by the water sample is probably due to some non-specific interactions between primers.

Question 27

Often there can be sample to sample differences during sample and nucleic acid preparation and cDNA synthesis. What can cause this?

Answer 27

Variations in initial sample amount.
Variations in nucleic and recovery.
Differences in sample and/or nucleic acid quality.
Variations in cDNA synthesis efficiency.

Question 28

_____ minimizes the effect of non-specific factors on the comparison of levels of specific nucleic acids between samples.

Answer 28

Normalisation.

Question 29

True or False: You can compare two research papers even if they used different normalization methods.

Answer 29

False- they have to have used the same normalization methods.

Question 30

What are the 2 characteristics of a qPCR test?

Answer 30

Analytical performance

Diagnostic performance

Question 31

How well the test can detect a target if it is present in the sample is known as _____ performance.

Answer 31

Analytical - you want the test to be highly sensitive, specific, linear over a broad range of target concentrations, precise and reproducible.

Question 32

_____ performance relates to the ability of the test to reliably distinguish between infected and non-infected animals. Linked to the prevalence of infection.

Answer 32

Diagnostic